Immunoglobulin recognition of synthetic and natural left-handed Z DNA conformations and sequences

Zarling, David A.; Arndt‐Jovin, Donna J.; Robert‐Nicoud, Michel; McIntosh, Lawrence P.; Thomae, Ralf; Jovin, Thomas M.

doi:10.1016/0022-2836(84)90495-9

Cited by 82 publications

(77 citation statements)

References 84 publications

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“…All sera were heat-inactivated for 15 min at 56°C, sodium azide was added to a final concentration of 0.05%, and aliquots were stored at -70°C. The anti-Br-poly[d(C-G)] IgG, designated T-4, was purified from serum as described (3). Letters refer to inoculated animals and bleeds are given as numberse.g., D-4 is rabbit D, fourth bleed.…”

Section: Methodsmentioning

confidence: 99%

“…Direct RIA or competition assays with unlabeled polymers or drugs were as described (3)(4)(5) Cytological Preparations, Immunofluorescent Staining, and Photomicroscopy. Crithidia luciliae and Crithidia fasciculata were propagated in culture medium containing 37 mg of brain-heart infusion per ml (Difco) and 25 ,ug of hemin per ml.…”

Section: Methodsmentioning

confidence: 99%

“…Z-DNA sequences exist in deproteinized negatively supercoiled plasmids, viral DNAs, and fixed (protein-depleted) cellular DNA (3,4). Nuclease cutting, topisomerase relaxation, or treatment with certain intercalating drugs can induce a Z --B transition in plasmid or cellular DNA and eliminate the binding of specific anti-Z-DNA IgG probes.…”

mentioning

confidence: 99%

“…Nuclease cutting, topisomerase relaxation, or treatment with certain intercalating drugs can induce a Z --B transition in plasmid or cellular DNA and eliminate the binding of specific anti-Z-DNA IgG probes. Small amounts of Z-DNA or potential Z-sequences can be detected by anti-Z-DNA IgG immunofluorescence in nuclear DNA from a variety of fixed cells (1)(2)(3)(4). Though it appears that some alternating purine-pyrimidine sequences can be transcribed in nuclear DNA, it is not known whether transcribed RNA can adopt the left-handed Z-RNA conformation.…”

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confidence: 99%

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Cytoplasmic Z-RNA.

Zarling¹,

Calhoun²,

Hardin³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgGs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Preor nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or singlestranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation.Alternating purine-pyrimidine sequences having a strong potential to adopt stable leftghanded Z-conformations (e.g., in negatively supercoiled DNAs) are repetitive elements in prokaryotic and eukaryotic DNA genomes. Some of these sequences are transcribed into RNA. The in vivo conformational states of these nucleic acids and their functions are not currently known (for reviews, see refs. 1 and 2).Z-DNA sequences exist in deproteinized negatively supercoiled plasmids, viral DNAs, and fixed (protein-deplet- A detailed report on the extent of bromine modification used to elicit the antibodies used here and its effect on their immunochemical specificities will be published elsewhere (C.C.H., D.A.Z., S. K. Wolk, W. Ross, and I. Tinoco, Jr., unpublished data). Polynucleotides were extensively dialyzed against TE buffer (10 mM Tris HCl, pH 7.4/0.1 mM EDTA). Polynucleotide kinase labeling was by the procedure of Silberklang et al. (6).Radioimmunoassay (RIA) and Nitrocellulose Filter Binding Assays. Direct RIA or competition assays with unlabeled polymers or drugs were as described (3-5). The standard RIA buffer was 200 mM NaCl/40 mM Tris HCl, pH 7.5/4 mM EDTA. Reaction mixtures (20 ,ul) containing 20 ng of polynucleotide substrate were equilibrated for 5 min at 37°C before adding 5 ,ul of the first antibody (either rabbit or mouse). Primary reaction mixtures were incubated for 1 hr at 37°C; this was followed by addition of 1 ,ul of the appropriate second antibody, either affinity-purified goat anti-rabbit IgG or goat anti-mouse IgG (2.4 mg/ml). After an additional hour at 37°C, immune complexes were washed (three times) with RIA buffer, centrifuged at 15,000 x g (10°C), resuspended, and then assayed for radioactivity in 5 ml of Aquasol-2 (New England Nuclear) in a liquid scintillation counter (Beckman). A machine background of 15-35 cpm was subtracted. Filter-binding assays were performed using Millititer nitrocellulose filters on a vacuum manifold (Millipore). Reactions were as described for RIA, except for the omission of the second antibody, and were terminated by the...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Cytoplasmic Z-RNA.

Zarling¹,

Calhoun²,

Hardin³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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“…Immunoglobulin fraction was purified from crude serum by ammonium sulfate precipitation (45% saturation) and chromatography on a DEAE-Sephadex A-50 column equilibrated in 50 mM sodium acetate/21 mM acetic acid, pH 5 [24]. IgG were collected in the column flow-through.…”

Section: Rabbit Immunization and Antibody Purificationmentioning

confidence: 99%

The Saccharomyces cerevisiae CDC25 gene product is a 180 kDa polypeptide and is associated with a membrane fraction

Garreau¹,

Camonis²,

Guitton

et al. 1990

FEBS Letters

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In the yeast Saccharomyces cerevisiae, the CDC25 gene product is supposed to interact with ras proteins and adenylate cyclase for progression through the cell division cycle. To identify the CDC25 gene product, we raised antibodies against two hybrid proteins, encoded by in-frame fusions between the E. coli IacZ gene and two different parts of the CDC25 gene. By protein immuno-blotting, we were able to identify the CDC25 gene product as a 180 kDa polypeptide, which we named ~180 cDc2s. It was detected only when the CDC25 gene was overexpressed in a proteases-deficient yeast strain. Subcellular fractionation experiments showed that p180cDczJ, as well as ras proteins, is attached to the membrane, even after treatments which release peripheral membrane proteins.

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Structure and Function of DNA

Götz¹

1992

Biotechnology

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Immunoglobulin recognition of synthetic and natural left-handed Z DNA conformations and sequences

Cited by 82 publications

References 84 publications

Cytoplasmic Z-RNA.

Cytoplasmic Z-RNA.

The Saccharomyces cerevisiae CDC25 gene product is a 180 kDa polypeptide and is associated with a membrane fraction

Structure and Function of DNA

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